The cellular basis of feeding-dependent body size plasticity in sea anemones

ABSTRACT Many animals share a lifelong capacity to adapt their growth rates and body sizes to changing environmental food supplies. However, the cellular and molecular basis underlying this plasticity remains only poorly understood. We therefore studied how the sea anemones Nematostella vectensis and Aiptasia (Exaiptasia pallida) respond to feeding and starvation. Combining quantifications of body size and cell numbers with mathematical modelling, we observed that growth and shrinkage rates in Nematostella are exponential, stereotypic and accompanied by dramatic changes in cell numbers. Notably, shrinkage rates, but not growth rates, are independent of body size. In the facultatively symbiotic Aiptasia, we show that growth and cell proliferation rates are dependent on the symbiotic state. On a cellular level, we found that >7% of all cells in Nematostella juveniles reversibly shift between S/G2/M and G1/G0 cell cycle phases when fed or starved, respectively. Furthermore, we demonstrate that polyp growth and cell proliferation are dependent on TOR signalling during feeding. Altogether, we provide a benchmark and resource for further investigating the nutritional regulation of body plasticity on multiple scales using the genetic toolkit available for Nematostella.


Fig. S9. Gating strategy in short EdU pulse (30-60min) experiments in
Nematostella (refers to experiments in Fig. 4I, J, Fig. 5C-E).In Nematostella short pulse EdU experiments, 7 animals constituted one biological sample and 3 biological samples were performed for each time point.We coupled incorporated EdU with Alexa488 fluophore azide (recorded in the FITC cytometer channel).1µg/ml FxCycle violet DNA dye (Invitrogen) was used to distinguish cell cycle phases (recorded in the "Pacific blue" cytometer channel 'DNA-A').We first excluded debris based on size and granularity in the FSC-A/SSC-A gate, with sub-gates based on FSC-A/FSC-H parameters and FSC-A/SSC-W parameters to remove potential cell doublets and high complexity events.We then gated particles based on DNA dye intensity in width over area and plotted a histogram of DNA dye in area on the linear scale to visualize the characteristic DNA dye intensity peaks expected from cells between 2N and 4N.
Based on the fluorescence signal of DMSO controls in the FITC-A channel within the 2N-4N pool of cells, a threshold was drawn above which cells were considered EdU+.

Fig. S11. Gating strategy in long (>24h) EdU pulse/chase experiments in
Nematostella (refers to experiments in Fig. 4K, L, S7).For the analysis of a long (>24h) EdU pulse, we excluded debris and gated cells based on DNA-dye intensity as explained above.However, we observed that the long-term incorporation of EdU interfered with the DNA stain fluorescence and prevented a clear separation of 2N-4N cells.We therefore split the EdU+ from the EdU-cell populations based on a DMSO negative control and assessed the cell cycle separately.In the EdU+ cells, we differentiated a population of G1/G0 events, with lower EdU/DNA fluorescence and a population of S/G2/M events with higher EdU/DNA signal.The cell cycle distribution of EdU-negative cell populations was defined by gating from a DNA-signal histogram.As expected, the level of S/G2/M cells in the EdU-negative population after a long EdU pulse was negligible.S3D: ANOVA for the effect of starvation day on the fraction of EdU positive cells and pairwise comparisons between days (Tukey's HSD) Table S3E.ANOVA for the effect of starvation day on the fraction of EdU positive cells and pairwise comparisons between days (Tukey's HSD) Table S3F.Flow cytometer analysis of long term EdU pulse (>24h) -cell cycle phases defined as per DNA content Table S3G.ANOVA for the effect of experimental treatment on the fraction of EdU+ cells and pairwise comparisons between treatments (Tukey's HSD) Table S3H.ANOVA for the effect of experimental treatment on the fraction of EdU+ S/G2M cells and pairwise comparisons between treatments (Tukey's HSD) Table S3I.ANOVA for the effect of experimental treatment on the log (FITC-A) intensity as a quantification of EdU incorporation in EdU positive cells and pairwise comparisons between treatments (Tukey's HSD) Table S3J.ANOVA for the effect of symbiotic state (symbiotic/aposymbiotic) on the fraction of EdU positive cells in Aiptasia and pairwise comparisons between treatments (Tukey's HSD)  S4H.ANOVA for the effect of DMSO (0.2%) or Rapamycin (4μM) treatment on median FSC-A (as proxy for cell size) Table S4A.Bodysize before and after 4 days of daily feeding under DMSO (0.2%) or Rapamycin (4μM) treatment Table S4B.EdU index before and after 4 days of daily feeding under DMSO (0.2%) or Rapamycin (4μM) treatment Table S4C.Cell cycle composition before and after 4 days of daily feeding under DMSO (0.2%) or Rapamycin (4μM) treatment Table S4D.Median FSC-A as a proxy for cell size before and after 4 days of daily feeding under DMSO (0.2%) or Rapamycin (4μM) treatment Table S4E.ANOVA for the effect of day (day 0 vs day 4) and treatment (DMSO (0.2%) vs Rapamycin (4μM) on bodysize Table S4F.ANOVA for the effect of DMSO (0.2%) or Rapamycin (4μM) treatment on the EdU index Table S4G.ANOVA for the effect of DMSO (0.2%) or Rapamycin (4μM) treatment on the fraction of cells in S-phase Available for download at https://journals.biologists.com/dev/article-lookup/doi/10.1242/dev.202926#supplementary-dataDevelopment • Supplementary information 3 Doubing times, half-lives and interval loss rates in starvation

Development • Supplementary information
We have generalyl observed that phenotypic variables in Nematostella vectensis undergo exponential growth and exponential decay of the form N (t) = N 0 e rt where r is the growth rate (if positive) or degrowth rate (if negative).These rates r of exponential behaviour are not necessarily the most interpretable way that this behaviour can be quantitatively reported.It is informative to compute the doubling times T d and the half-lives t 1/2 , giving the times that it takes for the initial quantity to double in number or to reduce to half, respectively.The corresponding expressions are, for doubling time, and for half-life,

Fig. S2 .
Fig. S2.Stereotypy of growth during feeding or re-feeding of Nematostella juveniles.(A-C) Body size (A,B) and log-transformed body size (C) of Nematostella juveniles starved for 170 (A) or 200 days (B) after 14 days of ad libitum feeding.In (A, B), means and 95% confidence intervals (c.i.) are connected across time points.At t0: n= 12 individuals per time point in both experiments (A, B). (C) Representation of linear regression model on the log transformed values.(D) Representation of growth rate estimates from fitting linear models to log-transformed body size values.Note overlap of slopes in animals fed ad libitum without prior starvation (see (1)-(2)) and of slopes from experiments with prior starvation (see 'phase 3' and 'refed' in (3) and (5)-(7)).(E, F) Length scales with the squared width in polyps fed ad libitum over 10 days (see also SI: Geometry).Body area measurements of the same individuals as shown in Fig. 3A.n=24 individuals per time point.c.i.: confidence interval; s.e.m.: standard error of mean.

Fig. S3 .
Fig. S3.Stereotypy of shrinkage during starvation in Nematostella juveniles.(A-J) Body size (A, F) and log-transformed body size (B-E, G-J) during 140 (A-E) or 200 (F-J) days of starvation.Black lines in (A, F) represent connected mean values and white overlay represents the 95% confidence interval per time point.At t0: n=90 individuals (A) or n=between 58 and 136 individuals from a larger pool (F).(B-E, G-J) Representations of multi-phased linear regression models for log-transformed body size during 140 days of starvation (B-E) or 200 days of starvation (G-J) with 100 bootstraps visualized as grey lines.Lowest Akaike Information Criterion (AIC) indicates best fit models highlighted by boxes (D, J; see SI). (K) Comparison of estimated slopes (in 95% confidence intervals (c.i.)) for all experiments on Nematostella shrinkage.Note that all slopes are narrowly distributed between approx.

Fig. S4 .
Fig. S4.Body size (A-D), cell number (E-H) and cell size (I-L) dynamics during 21 days of starvation in Nematostella juveniles.For 21 days starvation, multiphased linear regression models were fitted to the log-transformed values of body size, cell number and cell size from individual polyps.See also Fig. 3D, H, L. 100 bootstraps (in grey lines) were plotted to test 1 to 4 models.Best fitting models highlighted by blue boxes as based on Akaike Information Criterion (AIC; see SI).A model with one shrinkage phase (A, E, I) described body size, cell number and cell size changes best over 21 days of starvation.

Fig. S5 .
Fig. S5.Aiptasia polyps show feeding-dependent growth and shrinkage during starvation.(A-F) Multi-phased linear regression models were fitted to the logtransformed values of pedal disk area (see Fig. 1H) in aposymbiotic (A-C) and symbiotic (D-F) Aiptasia polyps.For both conditions, two phases best described the dynamics of size changes with a growth phase followed by a shrinkage phase.See SI for details on model selection.(G) Summary of slope values with 95% confidence interval for the best fitting change-point models.Note the overlap between growth slopes of symbiotic and aposymbiotic animals in 'phase 1'.During shrinkage in 'phase 2', shrinkage rates are higher in aposymbiotic than in symbiotic animals.(H) EdU index (60min pulse length) determined by flow cytometry at individual time points over the course of 21 days of starvation in aposymbiotic (APO; white) and symbiotic (SYM;

Fig. S6 .
Fig. S6.Validation of flow cytometry estimates for individual cell size and cell number.(A) Serial dilutions of animal homogenates were used to benchmark the cell counts obtained by flow cytometry.We found a near-perfect correlation (R² = 0.998) between the recorded cell count and the dilution coefficient.Each datapoint represents the mean of 2 technical replicates from a dilution of cells originating from a pool of 10 dissociated individuals.(B, C) We validated cell numbers obtained by flow cytometry analysis (see Fig. 3G) in a subset of the flow cytometry samples (n=10 replicates per timepoint) by Neubauer chamber-supported manual cell counting (B).We found a good correlation between the methods (C, R² = 0.798).(D) Mean recovery rates of 10µm yellow-green FluoSpheres added before tissue homogenization to estimate cell loss during dissociation plus s.e.m.Note that bead recovery was calculated per

Fig. S7 .
Fig. S7.Median fluorescent intensity measures of EdU label (B) and FxCycle violet DNA dye (C) points to a small population of cells proliferating feedingindependently. (A) Experimental setup of EdU pulse-chase experiments as in Fig. 4K-L.(B, C) Median EdU signal fluorescence intensity (B) and cell cycle distribution of EdU+ cells as estimated by EdU or FxCycle violet DNA dye signal intensity using flow cytometry (C).Statistical significance between pairwise comparisons was tested by Tukey's HSD post hoc test on one-way ANOVA analysis.***= p > 0.001 (see Table

Fig. S8 .
Fig. S8.Rapamycin treatment inhibits TOR signaling and changes cycle dynamics during feeding.(A) Western blots depict protein levels of Actin (as control), phosphorylated (p-RPS6) and unphosphorylated ribosomal protein S6 (RPS6) during feeding and 4 days of 0.2% DMSO or 4µM Rapamycin.For each sample, protein from pools of 50 juveniles was extracted.(B) Relative levels of p-RPS6 or RPS6 band intensities show that TOR inhibition using 4μM Rapamycin led to a decrease of global RPS6 and of p-RPS6 levels.(C, D) Representative confocal imaging stacks of juvenile midbody epidermis after 4 days of feeding and 0.2% DMSO or 4 μM Rapamycin treatment.Note that p-RPS6 (purple) and EdU signal (yellow; 30 min EdU pulse) were nearly abolished in Rapamycin-treated animals.Nuclei stained by Hoechst33342.(E-J) Representative plots of flow cytometry analysis of EdU-labeled cell suspensions(30min EdU pulse) from 4-days starved juveniles at T0 ('day0 untreated') and after 4 days of feeding and 0.2% DMSO, or 4µM Rapamycin treatment (see also Fig.5).Pools of 7-10 animals were dissociated for each of the biological replicates (n=3).The

Fig. S10 .
Fig. S10.Gating strategy to assess Nematostella cell numbers and cell sizes via cytometry (refers to experiments in Fig. 4J, S6F).In cell counting experiments, the number of cells per individual were estimated by the addition of a constant number of red fluorescent beads (10 μm FluoSpheres™ (580/605), Invitrogen; recorded in the PE channel).Based on the logic explained above, we excluded debris and gated cells within the cell cycle based on DNA-dye intensity.Using the ratio of counted beads to counted cells within the cell cycle allowed to back-calculate initial cell numbers per sample (see Materials and Methods).In the starvation experiment, a second set of yellow-green beads (10 μm FluoSpheres™ (505/515), Invitrogen; recorded in FITC channel) was added at a known concentration before dissociation and counted by cytometry to assess cell loss during dissociation.In addition, nonfluorescent polystyrene beads (with known diameter of 2.0 μm, 4.0 μm, 6.0 μm, 10.0 μm and 15.0 μm, Invitrogen) were used to create a reference of FSC-A values and allowed overlapping bead size-bins with the cells in the cell cycle gate to assess how cell size fractions change during starvation time points.

Fig. S12 .
Fig. S12.Gating strategy in short EdU pulse (60min) experiments in Aiptasia (refers to experiments in Fig. S5H).In Aiptasia short pulse EdU experiments, 3 polyps of the strain CC7 in either aposymbiotic and symbiotic state, constituted one biological sample and 3 biological samples were performed for each time point.For visualizing incorporated EdU we used Alexa488 fluophore azide (recorded in the FITC cytometer channel) and stained DNA with FxCycle violet (Invitrogen).For both symbiotic and apo-symbiotic animals, chlorophyll autofluorescence (recorded in PE channel) was used to distinguish symbiont-free Aiptasia cells from symbionts/symbiont-containing Aiptasia cells.EdU+ cells were gated based on DMSO negative controls above in the DNA signal range of S-phase cells.

Table S1A .
Slope estimates from multi-phase linear models for Nematostella and Aiptasia body size changes

Table S1B .
Changepoints in multi-phased linear models for Nematostella and Aiptasia body size changes

Table S1C .
Analysis of Nematostella growth / shrinkage rates in dependence of the individual starting size, phase (1-4) and feeding condition (RES vs AL)

Table S2A .
Summary of body size, cell number, cell size values during

Table S2B .
Changes in body size, cell number and cell size between timepoints

Table S2C .
Linear regression analysis of individual bodysize, cell number and cell size

Table S2D .
Linear model to estimate the effect of cell size or cell number on body size

Table S2E .
Linear model to estimate the combined effect of cell size and cell number on body size

Table S3A .
Fraction of EdU positive cells as counted by images from confocal microscopy

Table S3B .
Flow cytometer analysis after 60min of EdU pulse -cell cycle phases defined as per DNA content

Table S3C .
Comparison of the effect of the assay method (microscopy vs cytometry), day, and their interaction on the EdU index (ANOVA)